PROJECT SUMMARY The functions and connectivity of neurons are established and manifested by their constituent proteins. Monitoring the organization of individual proteins in specific neuronal subtypes in live brain tissue may therefore provide important readout of cellular and circuit properties underlying brain function. However, it remains challenging to visualize endogenous synaptic proteins in individual neurons in live tissue. Most studies rely on the overexpression of fluorescently tagged proteins of interest. However, protein overexpression can alter protein stoichiometry, trafficking, subcellular localization, and cell signaling, ultimately affecting cellular and circuit functions. Although `knock-in' strategies can in principle bypass protein overexpression, these strategies are rarely employed because typical knock-in approaches result in global expression of the labeled protein in all cells where the target protein is normally expressed. Global expression results in high fluorescence background and a lack of contrast in tissues, making high-resolution imaging difficult. To solve the above problems, we recently developed a novel genetic strategy called endogenous labeling via exon duplication (ENABLED). We have used this method to label the critical postsynaptic marker protein PSD-95 with the yellow fluorescent protein mVenus in all neurons, in a sparse subset of neurons, or in specific neuronal subtypes. Unlike the conventional approach to visualizing PSD-95, which involves PSD-95 overexpression, our strategy does not result in altered neuronal functions, and, for the first time, allows for the monitoring of PSD-95 at endogenous levels in individual neurons in living mice. Despite these advantages, the ENABLED strategy can be further optimized to broaden its applicability and enhance its sensitivity. Furthermore, to comprehensively examine neuronal functions and connectivity, multiple types of synaptic markers will need to be labeled. Here, we request funds to optimize the ENABLED strategy and use it to label the excitatory and inhibitory postsynaptic markers, PSD-95 and gephyrin, respectively, with 5-fold stronger signal in mice. We will also generate ENABLED mice in which PSD-95 can be labeled using different colors for dual-color analyses with other proteins. We will make the reagents available to the neuroscience community to provide fellow researchers with an unprecedented ability to monitor synaptic connectivity and plasticity under physiological conditions in brain slices and in behaving animals.